Enhanced Top-Down Protein Characterization with Electron Capture Dissociation and Cyclic Ion Mobility Spectrometry.
Enhanced Top-Down Protein Characterization with Electron Capture Dissociation and Cyclic Ion Mobility Spectrometry.
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DOI：
10.1021/acs.analchem.1c04870
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发表时间：
2022-03-08
影响因子：
7.4
通讯作者：
Voinov VG
中科院分区：
文献类型：
--
作者：
Shaw JB;Cooper-Shepherd DA;Hewitt D;Wildgoose JL;Beckman JS;Langridge JI;Voinov VG
变性的多电荷高质量蛋白质前体离子的串联质谱产生具有数百个不同电荷状态的宽且重叠的产物离子同位素分布的极其密集的光谱，其产生以前体离子为中心的未分辨“噪声”的升高的基线。质量分析仪和信号处理方法的发展，以提高质量分辨能力和前体和产品离子电荷通过溶液添加剂或离子-离子反应的操纵已被彻底探索作为解决方案的光谱拥塞。在这里，我们证明了电子捕获解离（ECD）与高分辨率循环离子迁移谱（cIMS）的效用，大大提高自上而下的蛋白质表征能力。使用ECD产物离子的cIMS和“迁移率分数”（即，2D迁移图片段的提取质谱（m/z与漂移时间））减少蛋白质ECD光谱的拥塞。对于小的蛋白质，如泛素（8.6 kDa），其中质量分辨能力不是表征的限制因素，前IMS ECD和迁移率分数没有显着增加蛋白质序列的覆盖率，但增加了识别的产物离子的数量进行了观察。然而，通过蛋白质序列覆盖率测量，观察到更大和更高电荷的物种，如碳酸酐酶（29 kDa）的+35电荷状态的性能急剧增加。与不使用IMS维度的处理相比，与迁移率分数结合的Pre-IMS ECD产生了注释同位素簇的数量增加135%和独特产物离子增加75%。这些结果产生碳酸酐酶的89%序列覆盖率。
Tandem mass spectrometry of denatured, multiply charged high mass protein precursor ions yield extremely dense spectra with hundreds of broad and overlapping product ion isotopic distributions of differing charge states that yield an elevated baseline of unresolved “noise” centered about the precursor ion. Development of mass analyzers and signal processing methods to increase mass resolving power and manipulation of precursor and product ion charge through solution additives or ion–ion reactions have been thoroughly explored as solutions to spectral congestion. Here, we demonstrate the utility of electron capture dissociation (ECD) coupled with high-resolution cyclic ion mobility spectrometry (cIMS) to greatly increase top-down protein characterization capabilities. Congestion of protein ECD spectra was reduced using cIMS of the ECD product ions and “mobility fractions”, that is, extracted mass spectra for segments of the 2D mobiligram (m/z versus drift time). For small proteins, such as ubiquitin (8.6 kDa), where mass resolving power was not the limiting factor for characterization, pre-IMS ECD and mobility fractions did not significantly increase protein sequence coverage, but an increase in the number of identified product ions was observed. However, a dramatic increase in performance, measured by protein sequence coverage, was observed for larger and more highly charged species, such as the +35 charge state of carbonic anhydrase (29 kDa). Pre-IMS ECD combined with mobility fractions yielded a 135% increase in the number of annotated isotope clusters and a 75% increase in unique product ions compared to processing without using the IMS dimension. These results yielded 89% sequence coverage for carbonic anhydrase.
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环状离子迁移率激活实验阐明了气相中的蛋白质行为。
DOI：
10.1021/jasms.1c00018
发表时间：
2021-06-02
影响因子：
3.2
作者：
Eldrid C;Ben-Younis A;Ujma J;Britt H;Cragnolini T;Kalfas S;Cooper-Shepherd D;Tomczyk N;Giles K;Morris M;Akter R;Raleigh D;Thalassinos K
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Thalassinos K
DOI：
10.1016/j.jasms.2009.01.005
发表时间：
2009-08-01
影响因子：
3.2
作者：
Makarov, Alexander;Denisov, Eduard;Lange, Oliver
通讯作者：
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影响因子：
7.4
作者：
Glaskin, Rebecca S.;Valentine, Stephen J.;Clemmer, David E.
通讯作者：
Clemmer, David E.
影响因子：
4.8
作者：
Lermyte, Frederik;Verschueren, Tim;Sobott, Frank
通讯作者：
Sobott, Frank
影响因子：
4.4
作者：
MARSHALL, AG
通讯作者：
MARSHALL, AG